Protein Z (PZ) is a vitamin K-dependent plasma protein whose function was previously unknown. Our work has shown that PZ serves as a cofactor to enhance (1000-fold) the inhibition of coagulation factor Xa bound at a phospholipid surface by a previously unidentified plasma protein called PZ-dependent protease inhibitor (ZPI). ZPI is a member of the SERPIN superfamily of protease inhibitors and not only inhibits factor Xa in a PZ-, Ca2+- and phospholipid-dependent manner, but also directly inhibits coagulation factor XIa. The aims of this proposal are to carefully characterize the biochemical mechanisms responsible for the actions of PZ and ZPI (Aim A) and to assess the physiologic relevance of the regulation of coagulation produced by PZ and ZPI through the use of human clinical materials (Aim B) and mouse models (Aim C). Experiments under Aim A will: 1) Define the kinetics of factor Xa and factor XIa inhibition by ZPI; 2) Characterize the interactions between factor Xa, PZ and ZPI at phospholipid surfaces; 3) Characterize the interaction between PZ and ZPI; and 4) Determine the structures within the gamma-carboxyglutamic acid domain of PZ and the amino-terminal extension in ZPI that may effect these functional properties. In Aim B, PZ and ZPI will be measured in a variety of populations/patient groups to determine the effect of age, gender, ethnic background, and hormonal therapy on their plasma levels and to evaluate their potential relationship to thrombotic disease. In Aim C, PZ and ZPI gene-deleted mice and mouse models of human disease will be used to explore the physiologic importance of these proteins.